A method for diagnosing TSE-induced changes in tissues using infrared spectroscopy.
1) Field of the Invention
This invention relates to a method for fast detection of pathologic changes induced by transmissible spongiform encephalopathies (TSE) in animal or human tissue using infrared spectroscopy (IR spectroscopy).
2) Brief Description of the Prior Art
Transmissible spongiform encephalopathies are communicable neurodegenerative diseases of the central nervous system (CNS) that may affect many mammals and humans. TSE is used as a cover term here that refers to the various forms of this disease as they occur in the various species. In addition to scrapie (trotting disease), the disease that originated in sheep but can be transmitted to hamsters and mice, five other types of TSE have become known as yet: Bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in some American deer and elk, transmissible mink encephalopathy (TME) in mink, feline spongiform encephalopathy (FSE) in cats, and a spongiform encephalopathy in antelopes. Four types of TSE are distinguished in humans: Creutzfeldt-Jakob disease (CJD), Gerstmann-Strxc3xa4ussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru.
TSE can definitely be diagnosed based on a) histological proof of characteristic spongiform changes in the brain tissue accompanied by gliosis, b) immunological proof of deposits of the pathologic prion protein (PrP) using a,Western blot test, histo-blot test, and immunohistochemistry, c) proof of scrapie-associated (PrP) fibrils (SAF) using an electron microscope, and d) proof of the infectious TSE agent using transmission experiments in animals.
Clinical symptoms and chemical laboratory findings of increased concentrations of specific proteins in cerebrospinal fluid and/or serum [Protein 14-3-3 (Zerr et al. (1997) N. Engl. J. Med. 336: 874; Zerr et al. (1998) Ann. Neurol. 43: 32-40.), Protein S100 (Otto et al. (1997) J. Neurol. 244: 566-570; Otto et al. (1998) Brit. Med. J. 316: S77S82; Otto et al. (1998) J. Neurovirol. 4: 572-573) as well as neuron-specific enolase (Zerr et al. (1995) Lancet 345: 1609-1610)] in animals and humans can only give rise to a tentative diagnosis. The same applies to changes visible in EEGs or MR tomograms that occur in conjunction with human TSE.
Development and improvement of detection procedures for TSE serve, inter alia, the following purposes.
a) Improving differential diagnostics of human TSEs. These diseases can only be diagnosed with any certainty by a post mortem or cerebral biopsy.
b) Detection of TSE contamination in blood, organs, and tissue and in products of human or animal origin produced from these.
c) Identification of blood, organ, and tissue donors infected with human TSE.
d) Detection of preclinical or clinical stages of TSE infection in farm animals (e.g. cattle and sheep) at the slaughterhouse or farm.
To be able to diagnose TSE diseases in farm animals is important because these diseases may be transmitted through eating the meat of diseased animals. It is suspected, for example, that the consumption of BSE-contaminated beef can cause a new variant of CJD in humans (nvCJD). Some states are currently introducing official monitoring of contamination levels in cattle populations to protect consumers and contain the spread of the epidemic. It is envisaged to carry out routine checks at slaughterhouses to establish whether the carcasses can be used.
Various test systems are being developed to provide sensitive and fast screening of large sample populations for pathologic prion protein and thus to provide diagnostics for large-scale production. These include a capillary electrophoresis immunoassay using fluorescence-labeled peptides (Schmerr and Jenny (1998) Electrophoresis 19: 409-419) and an immunological detection system using fluorescent lanthanide chelates called Delfia (Safar et, al. (1998) Nature Medicine 4. 1157-1165).
Only one diagnostic method is currently available to identify TSE-infected farm animals that is suitable for large-scale application. It is restricted to use at slaughterhouses and, according to the developer""s statements, can detect BSE in cattle up to half a year before clinical symptoms occur (see the manufacturer""s information on the Internet at http://www.prionics.ch).
In this method developed by Swiss-based Prionics AG, a tissue sample obtained from the medulla oblongata of slaughtered cattle is homogenized and treated with proteinase K enzyme. The pathologic prion protein that may remain after this treatment is labeled with the 6H4 monoclonal antibody (manufactured by Prionics) and then stained using the Western blot method. The manufacturer states that it takes up to 12 hours from taking the sample to getting a final result using this method. It is the problem of this invention to develop a method that provides fast, reliable and cost-efficient detection of TSE-induced tissue changes. The method according to the invention is to work efficiently in the routine operations of a slaughterhouse.
It is an object of this invention to provide a method for detecting TSE-induced pathologic changes in tissues. Said changes may be caused by scrapie, BSE, or any other of the TSE diseases. This objective is achieved by the present invention in the following steps: (a) directing infrared radiation onto a tissue sample and recording the spectral characteristics after interaction with the sample; (b) comparing the infrared spectrum thus obtained against a reference database containing infrared spectra of TSE-infected tissues and non-infected tissues; and (c) classifying the infrared spectrum as a spectrum obtained from TSE-infected or non-infected tissues.